This proposal is directed at gaining information of mitochondrial energy-transducing reactions directly involving the pyrophosphate bond. Further steady-state kinetic studies will be performed on ATP-drive reverse electron flow (succinate to NAD) catalyzed by heart submitochondrial particles using dead-end and product inhibitors to attempt to gain information about probable order of addition of reactants, departure of products, irreversible steps, etc. It is also proposed to carry out kinetic studies of mitochondrial ATPase from bovine heart mitochondrial vesicles, as well as other forms of non-membrane bound ATPase to attempt to correlate ATPase activity with the capacity for intermediate phosphate-water oxygen exchange capacity, with a view to determining the minimum structural requirement for this exchange. These studies may also throw some light on the nature of intermediates participating in the exchange and how they may be partitioned in various types of ATPase enzyme preparation depending on the position of rate limiting steps. The techniques employed range from spectrophotometric methods for kinetic studies to mass spectrometer studies for measurement of oxygen-18 exchange capacity. Data will be analyzed using various statistical programs by means of the Wayne State University IBM 360/67 computer. BIBLIOGRAPHIC REFERENCES: The existence of a 2,4 dinitrophenol-stimulated mitochondrial phosphate-water exchange and its implication for energy-coupling. R.A. Mitchell, C.M. Lamos, J.A. Russo (1975), 170th ACS National Meeting, Chicago, Ill., Abst. Biol. 107. Does a transition state give rise to an intermediate mitochondrial phosphate-water exchange characterized by resistance to uncouplers? R.A. Mitchell, C.M. Lamos, and J.A. Russo (1976) 20th Annual Meeting, Biophysical Soc., Seattle, Biophysical J. 16, 133a. (Abstract).